Method for the development of an HIV vaccine

ABSTRACT

Human immunodeficiency virus (HIV) comprising reverse transcriptase inactivated by photoinactivation used to evoke an immune response. The immune response may protect an individual from challenges with live virus. Alternatively, the inactivated HIV particles may be used to augment the immune response to HIV in an infected individual.

[0001] This application is a continuation-in-part application and claimspriority to prior pending provisional U.S. patent application No.60/074,646, filed on Feb. 13, 1998.

1.0 BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of diseasetreatment and prevention. More particularly, it concerns HIV particleswith inactivated reverse transcriptase and the use of such particles toelicit an effective immunological response to HIV. This immune responsewill provide protection from an HIV challenge and/or will assist theHIV-infected individual in controlling the replication of the virus.

[0004] 2. Description of Related Art

[0005] 1.2.1 Human Immunodeficiency Virus

[0006] Human Immunodeficiency Virus-1 (HIV-1) infection has beenreported throughout the world in both developed and developingcountries. HIV-2 infection is found predominately in West Africa,Portugal, and Brazil. It is estimated that as of 1990 there were between800,000 and 1.3 million individuals in the United States that wereinfected with HIV. An important obstacle to developing a vaccine againstHIV is that the mechanism of immunity to HIV infection isill-understood. Not all of those infected individuals will developacquired immunodeficiency syndrome (AIDS). Indeed recent reports havesuggested that there may be certain individuals that are resistant toHIV-1 infection.

[0007] The HIV viruses are members of the Retroviridae family and, moreparticularly, are classified within the Lentivirinae subfamily. Likenearly all other viruses, the replication cycles of members of theRetroviridae family, commonly known as the retroviruses, includeattachment to specific cell receptors, entry into cells, synthesis ofproteins and nucleic acids, assembly of progeny virus particles(virions), and release of progeny viruses from the cells. A uniqueaspect of retrovirus replication is the conversion of thesingle-stranded RNA genome into a double-stranded DNA molecule that mustintegrate into the genome of the host cell prior to the synthesis ofviral proteins and nucleic acids.

[0008] Retrovirus virions are enveloped and contain two copies of thegenome. The conversion of the genomic RNA into DNA is provided by theviral protein reverse transcriptase (RT). This protein is bound to theRNA genome within the virion, and its enzymatic conversion of the genometo DNA is believed to take place after viral entry into the host cell.However, recent evidence suggests that the conversion process mayinitiate in the virion particles themselves, known as endogenous reversetranscription (ERT), and that ERT may be important in increasing theinfectivity of the virus in sexual transmission (Zhang et al., 1993,1996).

[0009] Because of the requirement for reverse transcription in the viralreplication cycle, compounds that interfere with RT activity have beenutilized as anti-HIV therapeutic agents. Many of these compounds,including 3′-azido-2′,3′-dideoxythymidine (AZT), are nucleoside analogsthat, upon activation by host cell kinases, are competitive inhibitorsof reverse transcriptase (Furman et al., 1986). Other anti-RT compoundsare nonnucleoside inhibitors (NNI), hydrophobic compounds that do notrequire cellular modification for antiviral activity. Examples of suchcompounds include nevirapine (Grob et al., 1992; Merluzzi et al., 1990),the pyridinones (Carroll et al., 1993; Goldman et al., 1991), and thecarboxanilides (Bader et al., 1991; Balzarini et al., 1995, 1996). Thenevirapine analog9-azido-5,6-dihydro-11-ethyl-6-methyl-11H-pyrido[2,3-b][1,5]benzodiazepin-5-one(9-AN) and the carboxanilide analogN-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furanocarbothiamide(UC781™) have been shown to be potent inhibitors of RT. In respect ofthe 9-AN , the exposure of a mixture of this compound and RT toUV-irradiation has been particularly effective in inhibiting RT. Fromthe series of carboxanilides compounds, UC781™ has been found to beparticularly effective(Barnard et al., 1997). The addition of aphotoreactive label to UC781™ should increase further its ability toinactivate HIV RT, when a mixture of UC781™ and RT is exposed toUV-irradiation. The irradiation of a mixture of a photolabeled NNI of RTand RT is a type of photoinactivation.

[0010] The binding affinity and inhibitory effect of UC781™ is so highthat the compound was able to eliminate HIV infectivity following shortexposure of the isolated virus to UC781™ without the need forphotoinactivation (Borkow et al., 1997). Further, this compound wasshown to inhibit ERT in HIV virions and, when provided to HIV infectedcells, caused the production of noninfectious nascent virus (Borkow etal., 1997). Therefore, it appears that UC781™ is a particularly powerfulinactivator of HIV. Although UC781™ has been proposed for use inretrovirucidal formulations (Borkow et al., 1997), use as aphotoinactivator of HIV for the purpose of producing a vaccine is absentfrom the prior art.

[0011] 1.2.2 Immune Response to HIV

[0012] The immune response to HIV is composed of an initial cellmediated immune response followed by the subsequent development ofneutralizing antibodies. Within weeks of infection, virus titers in theblood fall coincident with the induction of anti-HIV cellular andhumoral immune responses. The fall in viremia correlates well with theappearance of anti-HIV major histocompatibility complex (MHC) classI-restricted CD8⁺ cytotoxic T cells (Haynes et al., 1996). Recentevidence has shown a strong correlation of anti-HIV CD4⁺ T cellresponses and reduced viral loads (Rosenberg et al., 1997). Therefore,the presentation of HIV antigens in the context of MHC class IImolecules to CD4⁺ T cells may be the key aspect of the control of theHIV infection.

[0013] Rosenberg et al (1997) suggest that in HIV-1 infection,HIV-specific CD4⁺ cells may be selectively eliminated. This may be dueto the activation of these cells during high-level viremia, increasingtheir susceptibility to infection (Weissman et al., 1996; Stanley etal., 1996), or may be due to activation induced cell death duringprimary infection (Abbas, 1996). Nonetheless, increasing thevirus-specific CD4⁺ T cell response without infecting, or destroying,the responding cells may be an effective means of controlling viralloads. Therefore, some existing HIV vaccines may be ineffective becausethey do not provide presentation of HIV peptides in the context of MHCclass II by antigen presenting cells.

[0014] 1.2.3 HIV Vaccines

[0015] Historically, viral vaccines have been enormously successful inthe prevention of infection by a particular virus. Therefore, when HIVwas first isolated, there was a great amount of optimism that an HIVvaccine would be developed quickly. However, this optimism quickly fadedbecause a number of unforeseen problems emerged. A discussion of theproblems that an HIV vaccine must overcome is provided within Stott andSchild (1996) and is incorporated herein by reference.

[0016] First, HIV is a retrovirus, thus, during its growth cycle,proviral DNA is integrated in the host genome. In this form the virus iseffectively protected from the immune response of the host and thisfeature of the virus suggests that effective vaccination must ideallyprevent the initial virus-cell interaction following transmission. Few,if any, of the currently available successful viral vaccines againstother infections achieve this level of protection. Secondly, HIVspecifically targets and destroys T-helper lymphocytes, which form anessential component of the immune response. Thirdly, the virus iscapable of extremely rapid antigenic variation which permits escape ofthe virus from immune responses. Fourthly, the majority of infectionsare acquired sexually via the genital or rectal mucosae, and infectionsof this route are generally considered difficult to prevent byvaccination. Finally, infection may be transmitted by virus-infectedcells in which the proviral DNA is integrated and viral antigens are notexpressed. Such a cell would not be recognized by immune responses toviral proteins and would therefore pass undetected. Few data areavailable to indicate how significant this mode of transmission is inthe overall epidemiology of HIV-1. Nevertheless, it represents apotential route and one which some authorities believe cannot be blockedby vaccination (Sabin, 1992).

[0017] Types of HIV vaccines include inactivated virus vaccines, liveattenuated virus vaccines, virus subunit vaccines, synthetic particlevaccines, and naked DNA vaccines and are reviewed in Stott and Schild(1996), Schultz (1996), and Johnston (1997). Several of these vaccinesare already in human trials.

[0018] The first evidence that vaccination against immunodeficiencyviruses was feasible came from early experiments using simpleinactivated virus prevented the onset of disease when vaccinated animalswere subsequently challenged (Desrosiers et al., 1989; Sutjipto et al.,1990). These results were confirmed and extended by Murphey-Corb et al(1989) who showed that most animals immunized with formalin-inactivatedvirus were protected against infection with SIV. Similar results weresubsequently obtained by several laboratories using virus-infected cells(Stott et al., 1990) or partially purified virus, inactivated byaldehydes (Putkonen et al., 1991, 1992; Johnson et al., 1992a; Le Grandet al., 1992), β-propiolactone (Stott et al., 1990) detergent (Osterhauset al., 1992) or psoralin and UV light (Carlson et al., 1990). Severaldifferent isolates of SIV or infectious molecular clones derived fromthem were used to prepare the vaccine and challenge viruses. A widevariety of adjuvants were also employed. On every occasion vaccinatedmacaques were protected against infection by intravenous challenge ofbetween 10-50 MID₅₀ (50% monkey infectious doses). Infections viruscould not be recovered from the blood or tissues of the protectedanimals even when they were followed for prolonged periods of over Iyear. Even more impressive was the failure to detect proviral DNA in thelymphocytes of protected animals, indicating that there had been nointegration of the challenge virus (Stott et al., 1990; Johnson et al.,1992a). It was thus clear that inactivated virus vaccines induced apowerful protective response in macaques. Unfortunately, the protectioninduced by inactivated SIV in macaques was not reproduced in chimpanzeesvaccinated with inactivated HIV and challenged with HIV-1 (Warren andDoltshahi, 1993).

[0019] 1.2.4 Photoinactivation of HIV

[0020] Methods of photoinactivation of HIV are known in the art and havebeen the subject of at least three patents. U.S. Pat. No. 5,041,078describes the use of sapphyrins in the photodynamic inactivation ofviruses, including HIV. U.S. Pat. Nos. 5,516,629 and 5,593,823 describethe use of psoralens and ultra violet light to inactivate HIV. U.S. Pat.No. 5,516,629 is incorporated herein by reference. Psoralens arenaturally occurring compounds which have been used therapeutically formillennia in Asia and Africa. A psoralen binds to nucleic acid doublehelices by intercalation between base pairs. Upon absorption of UVAphotons, the psoralen excited state has been shown to react with athymine or uracil double bond and covalently attach to both strands of anucleic acid helix. The crosslinking reaction is specific for a thymine(DNA) or uracil (RNA) base and will proceed only if the psoralen isintercalated in a site containing thymine or uracil. The initialphotoadduct can absorb a second UVA photon and react with a secondthymine or uracil on the opposing strand of the double helix tocrosslink the two strands of the double helix.

[0021] Lethal damage to a cell or virus occurs when a psoralenintercalated into a nucleic acid duplex in sites containing two thymines(or uracils) on opposing strands sequentially absorb 2 UVA photons. Thisis an inefficient process because two low probability events arerequired, the localization of the psoralen into sites with two thymines(or uracils) present and its sequential absorption of 2 UVA photons.

[0022] Attempts to inactivate viruses using photosensitizers and lighthave also been developed using some non-psoralen photosensitizers. Thephotosensitizers that have been employed are typically dyes. Examplesinclude dihematoporphyrin ether (DHE), Merocyanine 540 (MC540) andmethylene blue.

[0023] Carlson et al. (1990) has shown that a psoralen-inactivated wholeSIV (the Simian counterpart of HIV) vaccine can protect against lowchallenge doses of SIV and prevent early death in those monkeys that dobecome infected, suggesting that inactivated HIV may be an effectivevaccine in humans. However, because photoinactivation using psoralens isdependent on two rare events, a more efficient method of inactivation ispreferable to decrease the likelihood of live virus within a sample.Furthermore, these methods alter the antigenic conformation of HIVaffecting the production of an effective immunological response.

[0024] 3. Deficiencies in the Prior Art

[0025] Due to previous successes in preventing viral diseases usingsubunit, live-attenuated viral, and inactivated viral vaccines, thescientific community was initially optimistic that a vaccine would bedeveloped to prevent the spread of HIV. However, early optimism soondiminished because of repeated failures in the development of aneffective vaccine.

[0026] Subunit vaccines, although extremely safe, are limited in thebreadth of antigens that are presented to the immune system because onlyone or a few of the viral proteins are utilized in the vaccine. This maylimit the likelihood of cross protection between clades of HIV. Also,the production of subunit vaccines requires the molecular manipulationof the viral proteins into cloning or expression vectors, perhapsleading to increased production time and costs.

[0027] Live-attenuated HIV vaccines may also require molecularmanipulations in their production, although spontaneous attenuatedviruses may occur naturally. Attenuated HIV vaccines have includeddeletions in the nef region of the virus. Mutant-nef SIV vaccines showedinitial promise in primates, however, it was quickly shown that thesevaccines were capable of causing disease in newborn animals.Furthermore, recent evidence suggests that these vaccines are capable ofcausing full-blown AIDS in adult monkeys (Cohen, 1997). Therefore, thelack of an efficient understanding of HIV and its pathogenesis makes theuse of attenuated viruses a risky endeavor.

[0028] Inactivated viral vaccines provide a larger compliment ofantigens that are presented to the immune system, and, therefore,provide a greater amount of protection from HIV and is more likely toprovide protection across HIV clades. Furthermore, the inactivated viralvaccines do not require molecular manipulation of HIV and can be made toessentially any strain. Because inactivation of the virus is readilyshown in in vitro and animal models, the inactivated HIV vaccines areable to be tested in a timely manner to determine the effectiveness ofinactivation. Attenuated viruses may take years to determine theeffectiveness of the vaccine.

[0029] To be safe to administer to humans, efficient methods ofinactivation of HIV are required for vaccine production. Methods knownfor the inactivation include the use of aldehydes, β-propiolactone,psoralin and UV light, and others including detergents. Many of thesemethods alter the conformation of the virus thereby altering thespecificity of the immune response to the virus. Photoinactivation ofHIV using psoralin and UV light does not alter the conformation of thevirus, but it is an inefficient method of inactivation. Therefore, moreefficient methods of inactivation that do not affect the conformation ofthe virus would be ideal for use in the production of an HIV vaccine.

2.0 SUMMARY

[0030] The prior art is devoid of HIV vaccines created using anefficient method of inactivating HIV without causing deformation of theviral particle. The vaccine of the present invention is produced usingefficient methods of inactivation that do not alter the conformation ofthe virus to the same extent as other inactivation methods. Therefore,the vaccine of the present invention mimics infectious HIV particles butdoes not cause infection (i.e., establishment of a perpetuation of HIVwithin the recipient host due to incorporation of HIV into the genome ofcells within that host).

[0031] The use of an azido dipyrodiazepinona and an azidothiocarboxanilide (azido UC781™), azido-labeled compounds shown to bindand inactivate RT, permits the generation of non-infectious particles ofHIV by inactivating the HIV RT upon exposure of infectious particles toeither compound followed by irradiation with ultraviolet light. Theeffective inactivation of HIV RT by the methods described herein allowsthe production of non-infectious particles of HIV. These non-infectiousparticles of HIV have the capacity of eliciting an effective cellmediated and antibody mediated immune response which is protectiveagainst infection by HIV. The inactivated HIV particles of the presentinvention preserve the antigenic composition of infectious wild-type HIVparticles and thereby facilitate the dendritic cell-mediated processingand presentation of HIV particle-derived antigens to T cells.

[0032] The application of this methodology to different strains of HIVmay allow the production of a polyvalent vaccine (NIIPAV orNON-Infectious Immunogenic Polyvalent AIDS Vaccine). The inactivated HIVparticles of the present invention upon binding to CD4 receptors willexpose epitopes which may elicit broad immunogenic responses capable ofinhibiting the infectivity of diverse types of HIV from differentclades. Thus the exposure of the immune system to a single type ofinactivated particle of the present invention may protect againstinfection by multiple types of HIV.

[0033] One aspect of the present invention is a composition comprisingan HIV particle in which the RT is inactivated. The composition mayfurther comprise a pharmaceutically-acceptable excipient. The presentinvention contemplates that the HIV particle may be any type, subtype orisotype of HIV. In a preferred embodiment, the HIV particle is a wildtype HIV particle. In one embodiment the HIV particle is HIV 1. The HIV1 particle may be Group M or Group O. In different embodiments the GroupM HIV 1 may be clade A, clade B, clade C, clade D, clade E, clade F,clade G, clade H, or clade I. In a preferred embodiment of theinvention, the Group M particle is a clade B particle.

[0034] In one embodiment the RT is inactivated by one or more compoundsthat binds the RT and then irradiating bound RT with UV light. In apreferred embodiment the UV light is that emitted by a GE 275 W sunlamp. However, it is contemplated that any light that causes thereaction of the compound with RT may be used. In one embodiment of thecompound that binds to RT is an azido labeled compound. Essentially anyazido-labeled compound that binds to HIV RT and can penetrate the HIVparticle so as to associate with RT may be used. In a preferredembodiments the azido-labeled compound is azido dipyrodiazepinona orazido-UC781™. In other embodiments the azido-labeled compound is theazido derivative of 9-AN, UC38, UC84, UC10, UC82, UC040, HBY 097,calanolide A, or U-88204E. In one embodiment the inactivation of RTcomprises contacting said HIV particle with an effective amount of theazido labeled compound.

[0035] Another aspect of the present invention is a method of invokingan immune response in an animal by administering a compositioncomprising a pharmaceutically-acceptable excipient and an HIV particlein which the RT is inactivated. The immune response may be a humoralresponse, a cellular response or both a humoral and cellular responseThe cellular response may be a CD8+ T cell response, a CD4+ T cell, orboth a CD8+ T cell and a CD4+ T cell response.

[0036] The animal in which the immune response is invoked may be amammal. In preferred embodiments the mammal may be a human, a PBL-SCIDmouse, or a SCID-hu mouse. The animal in which the immune response isinvoked may be either HIV positive or HIV negative.

[0037] Another aspect of the present invention is a method of delayingthe onset of AIDS in an animal exposed to infectious HIV byadministering to the animal an inoculation of a pharmaceuticallyacceptable excipient and an HIV particle in which the RT is inactivated.The animal may be a mammal and in preferred embodiments the mammal is ahuman, a PBL-SCID mouse, or a SCID-hu mouse. The animal may be eitherHIV negative or HIV positive at the time of the administration of theinoculation.

[0038] Another aspect of the present invention is a method of making anHIV particle containing an inactivated RT comprising contacting acompound capable of inactivating RT with an HIV particle such that thecompound binds to the RT and then irradiating the HIV particle. In oneembodiment the HIV particle is HIV 1. The HIV 1 particle may be Group Mor Group O. In different embodiments the Group M HIV 1 may be clade A,clade B, clade C, clade D, clade E, clade F, clade G, clade H, or cladeI. In a preferred embodiment of the invention, the Group M particle is aclade B particle.

[0039] In one embodiment of the compound capable of inactivating RT isan azido labeled compound. Essentially any azido-labeled compound thatbinds to HIV RT and can penetrate the HIV particle so as to associatewith RT may be used. In preferred embodiments the azido-labeled compoundis azido dipyrodiazepinona or azido-UC781™. In other embodiments theazido-labeled compound is the azido derivative of 9-AN, UC38, UC84,UC10, UC82, UC040, HBY 097, calanolide A, or U-88204E.

[0040] Another aspect of the present invention is a method of preparinga composition comprising making an HIV particle containing aninactivated RT by contacting a compound capable of inactivating RT withan HIV particle such that the compound binds to the RT, irradiating theHIV particle, and then combining the HIV particle with the inactivatedRT with a pharmaceutically acceptable excipient.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0042]FIG. 1. Simplified phylogenetic tree of HIV-1. Group M denotes themajor group of HIV-1; group O refers to outliers or outgroup. Sequencedata from the env gene C2V3 region of selective isolates of subtypes Athrough H were compared to construct the phylogenetic tree. A ninthsubtype, I, has been reported recently but is not shown because theisolate was characterized using a different segment of the env gene. Thehorizontal branch lengths represent approximate relative geneticdistances. (From Hu D J, Dondero T J, Rayfield M A, et al., The emerginggenetic diversity of HIV: The importance of global surveillance fordiagnostics, research, and prevention. JAMA 275:210, 1996.)

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0043] The present invention relates to a vaccine comprisingRT-inactivated HIV for the purpose of eliciting a protective immuneresponse in an animal. The present invention employs methods ofinactivating RT. Further, the use of these inactivation methods for thepurpose of producing a vaccine is novel.

[0044] There are well described methods for the isolation and culture ofHIV. What is important to keep present in regard to the objectives ofthis invention is the fact that, using a methodology that inactivatesthe HIV reverse transcriptase, non-infectious particles of HIV areobtained which are capable of eliciting an effectively protective immuneresponse. Thus, although it makes sense to describe the method usingspecific strains of HIV and specific types of cells for the culture andtesting of the lack of infectiousness of the generated particles, itshould be kept in mind that this method may apply to different strainsof HIV, including laboratory and primary isolates.

[0045] In fact, given the geographic diversity of distribution ofdifferent strains of HIV, it makes sense to utilize many strains of HIVwith this methodology. The combined use of different strains ofinactivated particles of HIV is what will confer to this potentialvaccine preparation its polyvalent characteristics. Thus, in describingthe method to generate the vaccine particles and its testing for safetyand efficacy, the inventor sought to establish the general principles ofthe methodology for its subsequent application. It should be understoodthat the inactivator of the HIV reverse transcriptase to be used in theinitial study can be compared to other inactivators in parallel studiesand thus allow for the selection of the most effective one.

[0046] It of importance to note that the methodology of the presentinvention is applicable to any retrovirus which may be associated withany animal or human disease as a method for development of an effectivepreventive vaccine. Thus, the present invention has a broaderapplicability than the exemplified HIV vaccine.

[0047] 4.1 Human Immunodeficiency Virus

[0048] The genetic diversity of HIV is due to the extremely highreplication rate in infected individuals, the high rate of mutationcaused by the error-prone reverse transcriptase, the substantial viralload, and selection within infected individuals (Doolittle, 1989; Ho etal., 1995; Piatek et al., 1993). Diversity is so great that the presenceof closely related but not identical strains of HIV, known asquasispecies, commonly appear in a single, infected individual. Thequasispecies may diverge increasingly over time and changes tend to bewithin the env gene, particularly the V3 region (Hwang et al., 1992).Although changes also may occur in the gag, pol, and accessory genes,these differences tend to be less substantial.

[0049] When significant changes accumulate and are seen in a large groupof individuals, the strain is commonly considered a new family or newclade of HIV. Phylogenetic studies of HIV have shown that there are twomajor families of HIV, HIV-1 and HIV-2. Within the HIV-1 family thereare two major antigenic groups, known as Group M (major) and Group O(outlier). Each of these two groups has in turn different subtypes orclades which, when analyzed, lead to the conclusion that both probablyoriginated from two primordial viral ancestors. The group M isresponsible for most of the HIV infections throughout the world and thegroup O is rarely found and confined to Cameroon,Gabon and France. Thereare at least nine subtypes or clades in the group M and of these, thesubtype B is prevalent in the Western Hemisphere, while the subtypes A,C and D are in Africa. In Asia, the most frequently found subtypes areE, C and B, with the subtype E having a high prevalence in SoutheastAsia. In India the prevalent subtype is C. A phylogenetic tree based onthe sequence data from the env gene C2V2 region of selected isolates ofthe different subtypes is shown in FIG. 1. The geographic distributionof the different clades is shown in Table 1 (Hardy, 1996). TABLE 1Worldwide Geographic Distribution of HIV-1 Subtypes and HIV-2 HIV-1Subtypes Group M A B C D E F G H I O HIV-2 Africa + + + + + + + + + +Middle East + Europe + + + + + + + + + Asia + + + India + + + +Australia + North America + + South America + + +

[0050] A vaccine comprising one clade may provide for the protection ofinfection by one or more other clades. A very important concept whenconfronting what appears to be the very difficult challenge of antigenicvariation is the understanding of the concept of critical antigenicconsistency. By critical antigenic consistency it is meant that there isa critical number of epitopes which are found consistently in HIV.Although it is recognized that there are, significant antigenic changesin the configuration of the envelope proteins, generally, the internalproteins have less sequence variation. It has been recently demonstratedthat epitopes, of critical immunologic importance, are exposed orcreated as HIV begins to fuse with cell membranes. The fusion processresults in a conformational change of envelope glycoproteins leading tothe exposure of previously occult epitopes or the de novo formation ofepitopes. The recent use of these fusion exposed epitopes has led to thepreparation of antibodies which are capable of inhibiting theinfectivity of multiple primary HIV isolates, including multiple geneticsubtypes (Montefiori and Moore, 1999; LaCasse et al., 1999). The broadimmunological protection elicited by the fusion exposed epitopes mayexplain the observation that people infected with HIV-1 virtually neverhave more than one subtype of virus.

[0051] These significant recent results indicate that once the immunesystem is exposed to HIV without integration of HIV in the geneticmachinery of the host, the immune response will be effective and of abroad base. The non-infectious HIV particles of the part invention mimicthe antigenic structure and composition of natural infectious HIVparticles. Thus, these non-infectious particles will penetratesusceptible cells, including cells of the immune system responsible forthe generation of the immune response, in the identical fashion asinfectious particles, that is by receptor/co-receptor binding andfusion. The receptor-mediated entry of the vaccine into cells willresult in exposure of the superior immunogenic epitopes and therebyfacilitate the creation of a broad immunogenic response.

[0052] In addition to the recently described fusion exposed epitopes,the consistent regions of the env, gag, and pol together can lead to acritical mass of antigens responsible for the production of an effectiveimmunological response to HIV and, which in fact, are present in nearlyall types and subtypes of HIV. Thus, although it will be wise to usedifferent wild types to create non-infectious particles and create apolyvalent vaccine, it is also possible that exposure of the immunesystem to a single type of inactivated HIV particle will be enough togenerate a broad immune response.

[0053] The antigenic configuration of HIV is of the utmost importancesince it is known that conformational epitopes can be located invariable regions of the HIV particle and can not be predicted from theanalysis of the linear sequences of these regions. Therefore, it is ofgreat importance that, in eliciting an effective protective immuneresponse against HIV, the immune system is presented correct antigenicconformations.

[0054] Substantial evidence indicates that dendritic cells (“DC”)present in epithelial tissues (e.g., Langerhans cells) are the initialcells infected with HIV after mucosal exposure to the virus (Cameron etal., 1996; Knight, 1996). The bone marrow-derived DC are a class ofantigen-presenting cells (“APC”) that survey epithelial tissues foranitgens and are efficient stimulators of both B and T lymphocytes.Unlike B cells, T cells cannot directly recognize antigens and requirethat antigens be processed and presented by APCs (Banchereau andSteinman, 1998). Intracellular processing of antigens to peptidefragments results in binding to MHC class I molecules and a CD8+cytotoxic T cell response. In contrast, antigens that enter DC by theendocytic pathway generally bind to MHC class II molecules theelicitation of a CD4+ helper T cell response (Banchereau and Steinman,1998).

[0055] Inactivated HIV viral particles will be processed and presentedby DC as long as the inactivated HIV particles are preserved in itsantigenic composition and can access the cytoplasm of the dendriticcells. Both of these conditions are met by the present invention. Thatis, the inactivated particles have a preserved envelope structure andthus will access the cytoplasm of the dendritic cell by a process ofmicropinocytosis or mannose-receptor mediated uptake. DC that have beenexposed to the inactivated HIV particles will migrate to the lymph nodeswhere they will interact with T-cells presenting MHC-antigens complexesto both memory and naive T-cells (see Banchereau and Steinman, 1998;Bender et al., 1995). This process will lead to the development of aneffective anti-HIV MHC-I restricted CD8+ T-cell response. Cytotoxic CD8+T cells are recognized as having an important role in controlling HIVinvention (Musey et al., 1997; Oldstone, 1997).

[0056] Dendritic cells also have CD4/HIV co-receptors and thus can beinfected by HIV. This infectious process is independent of the captureand processing of HIV for antigenic presentation and initiation of theMHC class I restricted immunological response (Blauvelt et al., 1997).But since the inactivated particles are non-infections, the process ofpenetration through a receptor mechanism will allow the production of aMHC-II restricted response. Thus DC cells will activate and expand CD4+T helper cells, which in turn will induce B cell growth and antibodyproduction. This MHC class II response will thereby complement the MHCclass I restricted immune response by establishing an effectivecytotoxic and humoral response as well as an effective immunologicalmemory.

[0057] The inventor contemplates that the present invention may becomprised of inactivated viruses from one or more clades of HIV. Inpreferred embodiments, the vaccine may be comprised of inactivatedviruses of the clade or clades which with the individual is most likelyto come in contact. The data of Table 1 may be used as a guide todetermine which clades are prevalent in different geographical areas.

[0058] Because the present invention may be produced cheaply andrapidly, an individual may be vaccinated with inactivated virus or eveninactivated HIV infected cells from the individual most likely to passor have passed the virus to the individual. For example, an HIV-negativeperson may be vaccinated with inactivated HIV or inactivatedHIV-infected cells from an HIV-positive individual with which theHIV-negative individual plans to or has already come in sexual contact.An example of such a HIV-negative individual could be someone married toa hemophiliac that is HIV-positive. Additionally, these “personal”vaccines may have the benefit of also having cellular (nonviral) surfaceproteins from the individual passing the virus. The immune response tocellular surface proteins incorporated into the virus particles, whichinclude MHC antigens, have been shown to confer protection from futurechallenges from viruses grown in the same cell line (Stott et al.,1991).

[0059] 4.2 Photoinactivation of Reverse Transcriptase

[0060] A number of non-nucleoside inhibitors of HIV reversetranscriptase have been described and include neviprine and its analogs,the pyridobenzo- and dipyridodiazepinones, the pyridones, thequinoxalines, and the carboxanilides. Specific compounds include 9-AN,UC781™, UC38, UC84, UC10, UC82, UC040, HBY 097, calanolide A, U-88204E,and many others (Barnard et al., 1997; Esnouf et al., 1997; Buckheit etal., 1997; Kleim et al., 1997; Currens et al., 1996; Althaus et al.,1993). These compounds may be converted to azido photoaffinity labelsand utilized for the inactivation of HIV particles using methodsdescribed herein. The inventor contemplates that essentially anycompound that binds and inhibits HIV RT, is able to penetrate the viralparticle and associate with RT, and does not cause significantalterations in the conformation of the virus particle may be used toproduce an RT-inactivated virus for the purpose of eliciting aprotective immune response in an individual. Furthermore, the inventorcontemplates that the exposure of the photaffinity label-treatedparticles to light radiation to irreversibly inactivate the RT maycomprise of light of a variety of wavelengths. Although UV light,particularly that emitted by a GE 275 W sun lamp, is preferred, anyexposure to light that causes the reaction of the azido compound with RTis contemplated to be of utility in the production of the compositionsof the present invention.

[0061] 4.3 Vaccine Preparation

[0062] The inactivation of the virus by photoinactivation of RT providesnoninfectious, immunogenic particles that are essential identical inconformation and composition as infectious particles. Therefore, theinventor contemplates that particles inactivated in this method areideal for use as a potential vaccine against HIV diseases including AIDSand AIDS-related conditions. Thus the present invention provides animmunogenic composition that may be used as a vaccine against HIVinfection and its consequences, including AIDS and AIDS-relatedconditions. The immunogenic compositions elicit an immune response whichproduces cellular and humoral immune responses that are antiviral. If avaccinated person is challenged by HIV, T cells of the cellular responsewill eliminate infected cells and antibodies of the humoral responsewill inactivate the virus by binding to its surface.

[0063] Vaccines may be injectable liquid solutions or emulsions. TheRT-inactivated HIV particles may be mixed withpharmaceutically-acceptable excipients which are compatible with theinactivated virus particles. By compatible it is meant that thepharmaceutically-acceptable excipients will not alter the conformationalcharacteristics of the viral particle. Excipients may include water,saline, dextrose, glycerol, ethanol, or combinations thereof. Thevaccine may further contain auxiliary substances, such as wetting oremulsifying agents, buffering agents, or adjuvants to enhance theeffectiveness of the vaccines. Adjuvants may be mineral salts (e.g.,AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica, alum, Al(OH)₃, Ca₃(PO₄)₂,kaolin, or carbon), polynucleotides (e.g., poly IC or poly AU acids),and certain natural substances (e.g., wax D from Mycobacteriumtuberculosis, substances found in Corynebacterium parvum, Bordetellapertussis, or members of the genus Brucella) (PCT Application No.91/09603). Aluminum hydroxide or phosphate (alum) are commonly used at0.05 to 0.1 percent solution in phosphate buffered saline. Otheradjuvant compounds include QS21 or incomplete Freunds adjuvant.

[0064] Vaccines may be administered parenterally, by injectionsubcutaneously or intramuscularly, or the vaccines may be formulated anddelivered to evoke an immune response at the mucosal surfaces. Theimmunogenic composition may be administered to a mucosal surface by thenasal, oral, vaginal, or anal routes. The inventor contemplates that theadministration of the immunogenic compound to a mucosal surface that ismost likely to be challenged by HIV, such as the anal, vaginal, or oralmucosa, is preferred. For vaginal or anal delivery, suppositories may beused. Suppositories may comprise binders and carriers such aspolyalkalene glycols or triglycerides. Oral formulations may be in theform of pills, capsules, suspensions, tablets, or powders and includepharmaceutical grades of saccharine, cellulose or magnesium carbonate.These compositions may contain 10% to 95% of the RT-inactivated viralparticles.

[0065] Preferably the vaccines are administered in a manner and amountas to be therapeutically effective. That is to say that the vaccineshould be administered in such a way as to elicit an immune response tothe RT-inactivated viral particles. Suitable doses required to beadministered are readily discernible by those of skill in the art.Suitable methodologies for the initial administration and booster doses,if necessary, maybe variable also. The dosage of the vaccine may dependon the route of administration and may vary according to the size of thehost. One of skill in the art may obtain details regarding the practiceand use of the present invention in the American Foundation for AIDSResearch's HIV Experimental Vaccine Directory, Vol 1, No. 2, June 1998,which is hereby incorporated by reference in its entirety.

[0066] Although the immunogenic compositions of the present inventionmay be administered to individuals that are not infected with HIV,HIV-negative, they also may be administered to individuals who areinfected with the virus in an effort to alter the immune response to thevirus. The alteration may be a stimulation of anti-HIV CD4⁺ or CD8⁺ Tcells, an increase in antibody production, or in respect to the type ofresponse to the virus (i.e., T_(H)1 vs. T_(H)2). Nonetheless, thisalteration if effective will decrease the mortality and morbidityassociated with the HIV infection. In other words, the immunogeniccompound may decrease the severity of the disease and increase the lifeof the patient.

[0067] 4.4 Pharmaceutical Compositions

[0068] Where clinical application of a vaccine according to the presentinvention is contemplated, it will be necessary to prepare the complexas a pharmaceutical composition appropriate for the intendedapplication. Generally this will entail preparing a pharmaceuticalcomposition that is essentially free of pyrogens, as well as any otherimpurities that could be harmful to humans or animals. One also willgenerally desire to employ appropriate salts and buffers to render thecomplex stable and allow for complex uptake by target cells.

[0069] Aqueous compositions of the present invention comprise aneffective amount of the inactivated virus, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrases “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, or a human, as appropriate. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

[0070] Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0071] The inactivated viruses and inactivated virus-producing cells ofthe present invention may include classic pharmaceutical preparations.Administration of pharmaceutical compositions according to the presentinvention will be via any common route so long as the target tissue isavailable via that route. This includes oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration will be by orthotopic,intradermal, intraocular, subcutaneous, intramuscular, intraperitonealor intravenous injection. Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

[0072] The pharmaceutical compositions of the present invention areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified. A typicalcomposition for such purpose comprises a pharmaceutically acceptablecarrier. For instance, the composition may contain 10 mg, 25 mg, 50 mgor up to about 100 mg of human serum albumin per milliliter of phosphatebuffered saline. Other pharmaceutically acceptable carriers includeaqueous solutions, non-toxic excipients, including salts, preservatives,buffers and the like. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oil and injectable organic esterssuch as ethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components of the pharmaceuticalcomposition are adjusted according to well known parameters.

[0073] The compositions of the present invention may comprise asupplement of one or more compounds capable of preventing thereplication of HIV, including the compound utilized to inactivate thevirus. These compounds may include, but are not limited to, nucleosideanalog inhibitors of HIV RT (e.g., AZT), non-nucleoside inhibitors ofHIV-RT (e g., UC781™), or HIV protease inhibitors.

[0074] 4.5 Safety of the Vaccine

[0075] The safety of the vaccine particles may be demonstrated by theirinability to produce infection in susceptible cells regardless of theamount of particles used as inoculum. Controlled studies may conductedexposing susceptible cells to increased concentrations of theseparticles. Particles which have their RT inactivated will fail to infectsusceptible cells, while the control studies will maintain the capacityto produce infection in the susceptible cells. The same methodology thatwas used to generate the viral particles may be used to test theinactivation of the virus particles of the present invention. Formonitoring infectivity in both the non-infectious particles and thecontrols, the inventor contemplates the monitoring of production of RTand p24 antigen in the culture supernatants. In a preferred embodiment,supernatants are tested for the presence of virus particles by thesensitive method of heminested polymerase chain reaction (HNPCR)amplification of the 5′ LTR sequences (LTR-HNPCR). This test willconfirm the absence of infectivity of the vaccine particles since thereis an excellent correlation between a negative infectivity test and anegative LTR-HNPCR (Yang et al., 1998).

[0076] The safety of the particles can also be evaluated in vivo byinoculation of the animal models discussed infra in section 4.6. Thelack of infectivity of the inactivated particles can be determined byrepeated high dose inoculation of animals such as PBL-SCID mice, SCID-humice, or non-human primates.

[0077] As a way of creating an additional safety mechanism for thisvaccine, HIV integrase, an enzyme required for viral integration, can beinactivated. It is important to clarify that since the reversetranscritpase of the viral particle is inactivated there will be noreplication of the virus. The inactivated of HIV integrase would be anadded safety feature. Without a functional integrase there is nopossibility for the integration of HIV into the genetic material of thecell further ensuring the safety of the vaccine. The mechanism forintegrase inactivation will be one of selective photolabeling using a(as azido group) bound to any of several compounds that are known tobind to HIV-integrase. Among these compounds are: anti-integraseoilgonucelotides, L-chicoric acid, as well as a large number hydrazinederivative inhibitors.

[0078] 4.6 Administration

[0079] Although it is important to consider different routes ofadministration, the intramuscular route will be the route of choice.Other routes include: 1) intranasal; 2) intrarectal; 3) intravaginal; 4)oral and 4) subcutaneous. The dose to be used will be measured in viralparticles and it will have a range from the administration of 1 particleto 10²⁰ particles. It is anticipated that the optimal range of dosingwill be between 10⁴ particles and 10⁸ particles. Thus lower dose rangesmay include doses of about 10, 10², or 10³ particles. Optimal doseranges may include doses of about 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ particles.Higher dose ranges may include doses of about 10¹⁰, 10¹², 10¹⁴, 10¹⁶,10¹⁸ or 10²⁰ particles. The effective dosage may vary depending on themethod of administration.

[0080] For each dose to be tested, the schedule may consist ofadministration of a dose on days 0, 30, 60, and a booster dose at 180days. Alternatively doses may be given weekly, every two weeks, ormonthly for periods of one, two, three, four, five or six months. Dosesmay also be given every two months for a similar time. Periodic boostershots at intervals of 1-5 years may be desirable to maintain protectivelevels of immunity. Other administration schedules may be used and theinvention contemplates any administration schedule that results in aneffective vaccination.

[0081] In addition to monitoring for clinical safety, efficacy will beassessed by measuring the cellular and humoral immune response to HIV.Subjects will be followed for a period of two or more years from day 0(date of first inoculation).

[0082] 4.7 Animal Models

[0083] A number of different animal model systems for HIV infection havebeen employed (Kindt et al., 1992). Non-human primates such aschimpanzees and pig-tailed macaques can be infected by HIV-1. AlthoughCD4+ cells are not depleted in these systems, the animals are detectablyinfected by the virus and are useful in determining the efficacy of HIVvaccines. Small animal models include chimeric models that involve thetransplantation of human tissue into immunodeficient mice. One suchsystem is the hu-PBL-SCID mouse developed by Mosier et al. (1988)Another is the SCID-hu mouse developed by McCune et al. (1988). Of thetwo mouse models, the SCID-hu mouse is typically preferred because HIVinfection in these animals is more similar to that in humans. SCID-humice implanted with human intestine have been shown to be an in vivomodel of mucosal transmission of HIV (Gibbons et al., 1997). Methods ofconstructing mammals with human immune systems are described in U.S.Pat. Nos. 5,652,373, 5,698,767, and 5,709,843.

[0084] The animals will be inoculated with the vaccine of the presentinvention and later challenged with a dose of infectious virus. Efficacyof the vaccine will be determined by methods known by those of skill inthe art. Generally, a variety of parameters associated with HIVinfection may be tested and a comparison may be made between vaccinatedand non-vaccinated animals. Such parameters include viremia, detectionof integrated HIV in blood cells, loss of CD4+ cells, production of HIVparticles by PBMC, etc.. The vaccine will be considered effective ifthere is a significant reduction of signs of HIV infection in thevaccinated versus the non-vaccinated groups.

[0085] The ability of the inactivated HIV particles to elicitneutralizing antibodies can be measured in mice as previously described(LaCasse et al., 1999). The ability of sera to neutralize a range of HIVisolates can be tested using U87-CD4 cells expressing either CCR5 orCXCR4 coreceptors or by using an peripheral blood lymphocyte cultureassay (LaCasse et al., 1999, LaCasse et al., 1998; Follis et al., 1998).

[0086] 4.8 Application in Humans

[0087] Of course, the inventor contemplates the application of thepresent invention as a vaccine to HIV in humans. The inventorcontemplates that testing of the present invention as a vaccine inhumans will follow standard techniques and guidelines known by those ofskill in the art. One important aspect of human application is theproduction of an effective immune response to the vaccine. Althoughvarious ex vivo tests may be performed, such as measuring anti-HIVantibody production and anti-HIV cellular responses, the ultimate testis the ability of the vaccine to prevent infection by HIV or tosignificantly prolong the onset of AIDS in individuals receiving thevaccine. The monitoring of the efficacy of HIV vaccines in humans iswell known to those of skill in the art and the inventor does notcontemplate that the present invention would require the development ofnew methods of testing the efficacy of an HIV vaccine.

5.0 EXAMPLES

[0088] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

[0089] 5.1 Photoaffinity Labels

[0090] One compound that may be used to photoinactivate the reversetranscriptase of HIV-1, is an azido dipyridodiazepinona,9-Azido-5,6-dihydro-11-ethyl-6-methyl-11H-pyrido[2,3-b][1,5]benzodiazepin-5-one(9-AN). Production of 9-AN is described in Hargrave et al. (1991). Itmay be prepared by mixing an equimolar mixture of 2-chloronicotinic acidand 4-nitrophenylendiamine heated in sulfolane at 170° C. for 5 hours.After cooling, the precipitate is collected and washed with hot ethanol.The obtained mixture of 8- and9-nitro-5,6-dihydro-11H-pyrido[2,3-b]benzodizepin-5-ones, are thenmethylated with methyl iodide and dimsylsodium in DMSO, and the5,6-dihydro-6-methyl-9nitro isomer is purified by fractionalcrystallization. The 8-amino compound is obtained by ethylation withethyl iodide and dimsylsodium in DMSO followed by stannous chloridereduction of the nitro group. The 8-amino compound is then converted tothe azide by diazotization with sodium nitrate followed by reaction withsodium azide. The 9-azido derivative is then crystallized form diethylether to give analytically pure material: mp 115-118° C.; CIMS molecularion H* 295;C,H, and N analysis and spectroscopic characterization wereconsistent with the structure. A methanolic solution of the azidophotolabel has a lambda max of 248 nm(ε=30000).

[0091] Another molecule that can be prepared as a photolabel bydiazotization is a thiocarboxanilide, known by the trademark name ofUC781™. UC781™ was developed by Uniroyal Chemical Ltd. Researchlaboratories. It is known that UC781™ is a molecule with high affinityfor HIV-1 reverse transcriptase and with the characteristics of tightbinding. In fact, it is reasonable to assume that UC781™ aloneinactivates the HIV-1 reverse transcriptase (Barnard et al., 1997;Borkow et al., 1997). The diazotization of this molecule and itssubsequent activation with ultraviolet light further insures theinactivation of the HIV-1 reverse transcriptase. The azido photoaffinitylabels, upon exposure to ultraviolet light, transform into highlyreactive nitrenes capable of inserting into proximal covalent bonds ofthe HIV-1 reverse transcriptase enzyme structure.

5.2 Example 2 Inactivation of HIV Particles and HIV-Infected Cells

[0092] The inactivation of HIV can be accomplished by taking advantageof compounds that bind the HIV reverse transcriptase with a high degreeof specificity. Using the technique of photolabeling, a compound withhigh specific affinity for the HIV-1 reverse transcriptase can then beturned into an “active” moiety which will produce an irreversibleinactivation of the reverse transcriptase upon exposure to ultravioletlight irradiation. This inactivation, in the case of the compoundsalready described and known to have this effect, meets the requirementof being specific for the reverse transcriptase of HIV-1. That is, theirlabeling and photoactivation is not accompanied by the alteration of anyother component of the viral particle than the reverse transcriptase ofHIV-1. That is an important element of this invention since theirreversible inactivation of HIV-1 reverse transcriptase will lead tonon-infectious particles of HIV-1 with a natural antigenic structurewhich should behave as the infectious particles of HIV-1 insofar astheir capacity for stimulation of an effective immune response. Thereare many compounds that can be used for the purpose of photolabelinginactivation of the HIV-1 reverse transcriptase and include UC781™thiocarboxanilide, UC781™ azidothiocarboxanilide, and azidodipyridodiazepinona.

[0093] 5.2.1 Infection of Virus Producing Cells

[0094] Laboratory-adapted and primary wild type HIV isolates will becultured using established standard techniques. The cells to be used forculture will be phytohemagglutinin (PHA)-stimulated peripheral bloodmononuclear cells (PBMC's), since they are more readily infected by bothprimary isolates and laboratory-adapted isolates than cloned T-celllines (MT-2 or H9). Briefly, PBMC from normal blood donors are isolatedby Ficoll-Hystopaque gradient centrifugation and stimulated with 5 μg/mlof PHA for three to four days. The PHA-stimulated PBMC are then culturedin RPMI-1640 medium containing 10% heat-inactivated FBS, 100 Upenicillin/ml, 100 μg of streptomycin/ml 2 mM L-glutamine, and 5%purified human interleukin-2.

[0095] Aliquots of 10⁷ uninfected PBMC's in 10 ml of medium arepretreated with 2 μg/ml of polybrene for 1 h at 37° C. The cells arethen infected with a 1.0 ml inoculum of cell-free supernatant of theprimary isolate or the laboratory-adapted isolate. The infected PBMC'sare then resuspended in the culture media and monitored for supernatantp24 antigen concentration, which generally peaks at day 14postinoculation. The culture supernatants are harvested, pooled, andclarified through a 0.45 μm filter, and aliquoted. The 50% tissueculture infectious dose is determined according to the protocoldescribed by Johnson et al., (1990) using the HIV p24 antigen detectiontechnique.

[0096] Of course, the inventor contemplates that the use of PBMCs maynot be feasible when large volumes of virus are needed. In thisinstance, the cell line utilized is MT-2 grown in RPMI 1640 medium with10% heat-inactivated fetal calf serum (FBS), glutamine and antibiotics.Cells are propagated at 37° C. in an atmosphere of 5% CO₂ in air. Thevirus employed for this work is HIV-1 isolates IIIB and/or RF, which areprepared by an acute infection process. Virus infection of the MT-2cells is carried out in a bulk infection process. The appropriate numberof cells is mixed with infectious virus in a conical centrifuge tube ina small total volume of 1-2 milliliters. Following a 4-hour incubation,the infected cells are brought to the appropriate final concentration of5×10⁴ cells per milliliter with fresh tissue culture medium. Uninfectedcells at the same concentration are plated for the toxicity controls andfor the cell controls. The MOI is adjusted to give complete cell killingin the virus control wells by Day 6. Virus particles are concentratedusing standard techniques and quantified using RT assays (Fletcher etal., 1995a, 1995b) and p24 antigen assays.

[0097] 5.2.2 HIV Particle Inactivation

[0098] Once the HIV particles are purified and quantified, the 50%tissue culture infective dose ([TCID₅₀]=5×10⁴) is incubated in thepresence of four to eight times the 50% inhibitory concentration (IC₅₀)of the photoaffinity labeling molecule. In the case of the azidodipyridodiazepinona, the IC₅₀ is 160 nM. For the thiocarboxanilide andthe azido thiocarboxanilide, the IC₅₀ is 0.2 nM. Incubations were inRPMI 1640 without FBS for 2 h at 37° C. with gentle agitation every 15min. The mixtures of viral particles and photoaffinity labels areexposed to ultraviolet light using a GE 275-W sun lamp that provides aUV-irradiation intensity of about 15 μW/cm² for a period of at least 50minutes. After this process, the solution contains particles of HIV-1with a completely inactivated reverse transcriptase and thus unable toinfect susceptible cells.

[0099] The infectivity of the inactivated virus particles will bedetermined by controlled experiments where exposure of susceptible cellsto increased concentrations of these particles will fail to produceinfection of the cells as evidenced by the sensitive heminested PCRtechnique described in Yang et al. (1998) and the lack of production ofviral particles, reverse transcriptase, and p24. Such a process to assaythe production of virus particles is outlined by Borkow et al. (1997).Briefly, 0.5 ml of concentrated inactivated virus is added to 0.5 ml ofphytohemagglutinin-activated cord-blood mononuclear cells (CBMC)(4×10⁶cells) in RPMI 1640-10% FBS and incubated for 2 h at 37° C. withoccasional gentle mixing. The HIV-CBMC incubation mix is diluted withthe addition of 10 ml of RPMI 1640, and residual HIV is removed bypelleting the cells at 300×g for 10 min, followed by removal of thesupernatant and resuspension of the cells in 2 ml of RPMI 1640-10% FBScontaining interleukin-2 (10 U/ml). The entire sample is plated into asingle well of a 24-well dish. After 4 days of culture, 1 ml of mediumis removed and replaced with 1 ml of fresh medium. On day 7, culturesupernatants are isolated and HIV production is assessed by themeasurement of RT activity and p24 antigen levels in these cell-freesupernatants and cells are be monitored for integration by theheminested PCR technique of Yang et al. (1998).

5.3 Example 3 Production of Noninfectious Nascent Virus fromUC781™-Treated HIV Infected Cells

[0100] In addition to the inactivation of purified virus, UC781™ hasbeen shown to inactivate nascent virus from HIV-infected cells grown inthe presence of the compound (Borkow et al., 1997, incorporated whereinby reference). The methods described by Borkow et al. may be used toproduce inactivated virus for use in the present invention. Furthermore,the methods may be used to create a whole-cell vaccine in which thecells are HIV infected but rendered noninfectious by UC781™. A wholecell vaccine comprises the injection of HIV-infected cells. Theinjection of whole cells may provide a more vigorous immune response tothe virus. The methods of Borkow et al. (1997) are described below.

[0101] 5.3.1 Incubation of Chronically HIV-1 Infected H9 Cells withUC781™

[0102] Chronically infected H9 cells (5×10⁵ cells) are incubated with 10μM of UC781™ in a total volume of 1 ml of RPMI 1640-10% FBS for 18 h at37° C. The cells are then separated from the culture supernatants bycentrifugation at 300×g for 10 min. The pelleted H9 cells are washed bysuspension in 10 ml of RPMI 1640 followed by centrifugation at 300×g for10 min. The cell pellet is resuspended in 4 ml of RPMI 1640-10% FBS andused in coculture experiments to determine infectivity (Borkow et al.,1997).

[0103] 5.3.2 Incubation of Peripheral Blood Lymphocytes with UC781™

[0104] Peripheral blood lymphocyte (PBL) cells (2×10⁶ cells) isolatedfrom blood of HIV-1-infected patients are incubated with medium and 10μM UC781™ in a total volume of 1 ml for 2 h at 37° C. Excess drug may beremoved by pelleting the cells by centrifugation at 300×g for 10 min andremoval of the medium. The cell pellet is washed by suspension in 10 mlof medium followed by centrifugation. This washing step is repeatedtwice. The final cell pellet is resuspended in 1 ml of medium or anotherisotonic solution. To insure that the cells are noninfectious, they arecocultured with 1 ml of activated CBMC (2×10⁶ cells). The culture mediumis changed every 2 days, and fresh activated CBMC (2×10⁶ cells) areadded once per week. HIV-1 production is monitored by measurement of p24antigen levels in cell-free culture supernatants. Integration of thevirus is tested by the heminested PCR technique of Yang et al. (1998).

5.3 Example 3 Clinical Trail for HIV Vaccine

[0105] This example describes a protocol to facilitate an HIV vaccineclinical trial. The various elements of conducting a clinical trial,including patient treatment and monitoring, will be known to those ofskill in the art in light of the present disclosure. Generally, theclinical study of the vaccine composed of inactivated viral particlesshould consist of the administration of such viral particles produced bythe photolabeling of reverse transcriptase, as described in the presentinvention, to human subjects to evaluate safety and cellular, antibody,humoral and other clinical responses. The following information is beingpresented as a general guideline for use in HIV vaccine clinical trials.Information regarding design of clinical trials can also be obtained inthe American Foundation for AIDS Research's HIV Experimental VaccineDirectory, Vol 1, No. 2, June 1998.

[0106] 5.3.1 Eligible Subjects

[0107] Adult males and females HIV seronegatives.

[0108] 5.3.2 Subjects Inclusion Criteria

[0109] Patient Age: 18 years-60 years.

[0110] 5.3.4 Reproductive Criteria

[0111] Negative pregnancy test. Abstinence or effective method of birthcontrol/contraception during the study.

[0112] 5.3.5 Inclusion Criteria

[0113] The subject must be healthy as defined by a normal physical examand normal laboratory parameters as defined by the WHO for participantsin clinical studies. Subjects must be able to understand and sign aninformed consent. Subjects must also have a normal total white bloodcell count, lymphocyte, granulocyte and platelet count as wellhemoglobin and hematocrit. Subjects must has normal values of thefollowing parameters: urinalysis; BUN; creatinine; bilirubin; SGOT;SGPT; alkaline phosphatase; calcium; glucose; CPK; CD4+ cell count; andnormal serum immunoglobulin profile.

[0114] 5.3.6 Exclusion

[0115] The following are exclusion criteria: HIV-seropositive status;Active drug or alcohol abuse; inability to give an informed consent;medication which may affect immune function with the exception of lowdose of nonprescription-strength NSAIDS, aspirin, or acetaminophen foracute conditions such as headache or trauma; any condition which in theopinion of the principal investigator, might interfere with completionof the study or evaluation of the results.

[0116] 5.3.7 Randomization

[0117] The study will be double blind randomized. The placebo will bethe vaccine solution without the inactivated viral particles. Subjectswill be assigned randomly to one of the vaccine routes described above.

[0118] 5.3.8 Dose Range

[0119] Doses of 10⁴, 10⁶ and 10⁸ particles will be studied for clinicalsafety and immunogenicity. Other does in the range of 10 to 10²⁰,particles may also be studied.

[0120] 5.3.10 Administration

[0121] For each dose to be tested, the schedule may consist ofadministration of a dose on days 0, 30, 60, and a booster dose at 180days. Route of administration will be intramuscular. Additional routesof administration may include: subcutaneous; oral; intrarectal;intravaginal; intranasal/intramuscular; intrarectal/intramuscular;intranasal/subcutaneous; intrarectal/subcutaneous

[0122] 5.3.11 Number of Subjects Per Route of Administration

[0123] There will be 12 subjects per route of administration per doselevel. Of the 12 subjects 8 will receive the vaccine and 4 will receivea solution without inactivated viral particles.

[0124] 5.3.12 Duration of the Study

[0125] 24 months.

[0126] 5.3.13 Endpoints

[0127] The endpoint for clinical safety is no evidence of alteration ofthe clinical, immunological or laboratory parameters. The endpoint forimmunological efficacy is seroconversion with production of an effectivecellular, humoral and antibody response against HIV. The effectiveimmunological cellular response can be studied by using cytotoxic Tlymphocytes responses against different clashes of HIV. The humoralresponse can be evaluated by measuring the production of IFN-gammarelease using a modified Elispot assay. The antibody production can beassessed by performing neutralization studies against different cladesof HIV.

[0128] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

[0129] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0130] U.S. Pat. No. 5,041,078.

[0131] U.S. Pat. No. 5,516,629.

[0132] U.S. Pat. No. 5,593,823.

[0133] U.S. Pat. No. 5,652,373.

[0134] U.S. Pat. No. 5,698,767.

[0135] U.S. Pat. No. 5,709,843.

[0136] PCT Application No. 91/09603.

[0137] Abbas, Cell, 84:655, 1996.

[0138] Althaus et al., Biochemistry, 32(26):6548-6554, 1993.

[0139] American Foundation for AIDS Research's HIV Experimental VaccineDirectory, Vol 1, No. 2, June 1998.

[0140] Bader, McMahon, Schulz, Narayanan, Pierce, Weislow, Midelfort,Stinson, Boyd, Proc. Natl. Acad. Sci. U.S.A., 88:6740-6744, 1991.

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What is claimed is:
 1. A composition comprising an HIV particlecomprising inactivated reverse transcriptase.
 2. The composition ofclaim 1, further comprising a pharmaceutically-acceptable excipient. 3.The composition of claim 1, wherein said HIV particle is HIV-1.
 4. Thecomposition of claim 3, wherein said HIV-1 is Group M or Group O.
 5. Thecomposition of claim 4, wherein said Group M are selected from the groupconsisting of clade A, clade B, clade C, clade D, clade E, clade F,clade G, clade H, and clade I.
 6. The composition of claim 4, whereinsaid Group M particles are clade B particles.
 7. The composition ofclaim 1, wherein said reverse transcriptase has been inactivated viabinding said reverse transcriptase with one or more compounds that bindssaid reverse transcriptase and irradiating said HIV particles comprisingreverse transcriptase bound by said one or more compounds with UV light.8. The composition of claim 7, wherein said binding of said reversetranscriptase with one or more compounds is irreversible.
 9. Thecomposition of claim 7, wherein said compounds are azido-labeledcompounds.
 10. The composition of claim 9, wherein said azido-labeledcompound is azido dipyrodiazepinona or azido-UC781™.
 11. The compositionof claim 10, wherein said azido-labeled compound is azido-UC781™. 12.The composition of claim 7, wherein said inactivation comprisescontacting said HIV particle with an effective amount of UC781™.
 13. Amethod of invoking an immune response in an animal which comprisesadministering to said animal a composition comprising apharmaceutically-acceptable excipient and an HIV particle comprisinginactivated reverse transcriptase.
 14. The method of claim 13, whereinsaid immune response is a cellular response
 15. The method of claim 13,wherein said immune response is a humoral response.
 16. The method ofclaim 15, wherein said cellular response comprises CD8+ T cells.
 17. Themethod of claim 15, wherein said cellular response comprises CD4+ Tcells.
 18. The method of claim 13, wherein said animal is a mammal. 19.The method of claim 18, wherein said mammal is a PBL-SCID mouse or aSCID-hu mouse.
 20. The method of claim 18, wherein said mammal is human.21. The method of claim 13, wherein said animal is HIV-negative.
 22. Themethod of claim 13, wherein said animal is HIV-positive.
 23. A method ofdelaying the onset of AIDS in an animal exposed to infectious HIV whichcomprises administering to said animal one or more inoculations of thecomposition of claim
 1. 24. The method of claim 23, wherein said animalis a mammal.
 25. The method of claim 24, wherein said mammal is aPBL-SCID mouse or a SCID-hu mouse.
 26. The method of claim 24, whereinsaid mammal is a human.
 27. The method of claim 23, wherein said animalis HIV-negative at the time of administration of the composition ofclaim
 1. 28. The method of claim 23, wherein said animal is HIV-positiveat the time of administration of the composition of claim
 1. 29. Amethod of making an HIV particle comprising an inactive reversetranscriptase comprising: a) obtaining an HIV particle comprisingreverse transcriptase; b) obtaining a compound capable of bindingreverse transcriptase; c) contacting said HIV particle with saidcompound such that said compound binds said reverse transcriptase; d)irradiating said HIV particle
 30. The method of claim 29, wherein saidHIV particle is HIV-1.
 31. The method of claim 30, wherein said HIV-1 isGroup M or Group O.
 32. The method of claim 31, wherein said Group M areselected from the group consisting of clade A, clade B, clade C, cladeD, clade E, clade F, clade G, clade H, and clade I.
 33. The method ofclaim 31, wherein said Group M particles are lade B particles.
 34. Themethod of claim 29, wherein said compound is an azido-labeled compound.35. The method of claim 34, wherein said azido-labeled compound is azidodipyrodiazepinona or azido-UC781™.
 36. The composition of claim 35,wherein said azido-labeled compound is azido-UC781™.
 37. A method ofpreparing a composition comprising: a) obtaining an HIV particlecomprising an inactive reverse transcriptase comprising: i) obtaining anHIV particle comprising reverse transcriptase; ii) obtaining a compoundcapable of binding reverse transcriptase; iii) contacting said HIVparticle with said compound such that said compound binds said reversetranscriptase; and iv) irradiating said HIV particle; b) combining saidparticle into a pharmaceutically acceptable excipient.